Addiction to psychostimulants such as Methamphetamine (MA) is a significant public health issue in the United States and currently there are no FDA approved pharmacological interventions. Psychostimulant addiction is a heritable substance use disorder, however, its genetic basis is almost entirely unknown. Genome-wide association studies (GWAS) in humans currently lack sufficient power to detect the influence of common genetic variation on psychostimulant addiction. Mammalian model organisms offer an attractive alternative to more rapidly uncover novel genetic factors that contribute to addiction-relevant neurobehavioral traits. Using quantitative trait locus (QTL) mapping in mice, we identified a locus on chromosome 11 that caused a decrease in sensitivity to the locomotor stimulant properties of MA. To fine map this QTL, we generated interval-specific congenic lines and deduced a 206 kb critical interval on chromosome 11 that contained only two protein coding genes (Rufy1 and Hnrnph1). Replicate mouse lines heterozygous for Transcription Activator-like Effector Nucleases (TALENs)-induced frameshift deletions in Hnrnph1 (Hnrnph1+/-), but not in Rufy1 (Rufy1+/-), recapitulated the decrease in MA sensitivity observed in congenic mice, thus identifying Hnrnph1 as a novel quantitative trait gene for MA sensitivity. Hnrnph1 has not previously been identified in human GWAS of neuropsychiatric disorders but has been implicated in mu-opioid receptor splicing associated with heroin dependence. The primary objective of this proposal is to identify the functional mechanisms by which Hnrnph1 regulates MA addictive behaviors. RNA-sequencing and transcriptome analysis of the striatum suggests that inheritance of the QTL affects mesolimbic dopaminergic neuron development and striatal neurotransmission. Moreover, preliminary data extends the role of Hnrnph1 to the rewarding properties of MA as measured in the conditioned place preference (CPP) paradigm. To further investigate addiction-relevant behaviors in Hnrnph1+/- mice, in Aim 1 we will assess dose-dependent conditioned MA reward via MA-CPP as well as MA reinforcement via operant oral self-administration. In Aim 2, we will then identify the mesolimbic transcriptome, gene networks, spliceome, and the predicted HNRNPH1 targetome in the ventral midbrain and ventral striatum of Hnrnph1+/- mice. In Aim 3, we will use immunohistochemical staining of tyrosine hydroxylase to identify changes in ventral midbrain dopaminergic cell body density and presynaptic striatal innervation following heterozygous Hnrnph1 deletion. Additionally, we will determine the degree of co-localization between HNRNPH1 and D1 versus D2-type dopamine receptors in medium spiny neurons of the striatum. Finally, we will utilize in vivo microdialysis to measure baseline and MA-induced changes in striatal dopamine release in Hnrnph1+/- mice. These results will provide novel genomic and functional insights into Hnrnph1 regulation of mesolimibic circuitry function, MA addictive behaviors, and potentially inform upon novel targets for prevention and treatment of psychostimulant addiction.